Production of saliva is critical for the protection and maintenance of the hard and soft tissues of the mouth. Analysis of salivary gland function and the processes which regulate the coordinated transcription of a set of genes in the salivary gland has been limited by the lack of cell lines which maintain the differentiated phenotype. We propose to develop mouse parotid gland cell lines by adapting parotid acinar cells to continuous culture. Transgenic mice expressing both the transforming oncogene SV40 T antigen and the neomycin gene, will be generated in order to provide a continuous source of transformed parotid cells for establishing cell lines. The parotid specific transcriptional control region of the human salivary amylase gene will be used to direct expression of T antigen and neomycin to parotid acinar cells in the transgenic mice. Cell lines will be established from neoplastic issue, using the antibiotic G418 as a positive selection for parotid cells which maintain some differentiated phenotypes, since neomycin expression is dependent on transcription of the amylase promoter. Cell lines will be analyzed for expression of a number of parotid specific genes to determine if their transcriptional properties mimic parotid issue. Established parotid lines will then be used to elucidate the mechanism of salivary amylase gene expression. Transfection of amylase gene constructs into these cells will be carried out in order to map the location of cis-acting parotid enhancer elements. Nuclear transacting factors which interact with these cis-acting elements will be analyzed by gel mobility shift assays and DNase I footprinting experiments. Characterization of the mechanism of salivary amylase expression will provide a better understanding of the process of coordinated expression of a set of parotid-specific genes which are critically important for proper salivary function. The differentiated acinar cell lines developed in these studies will be a valuable resource for investigators studying the regulation of salivary cell function.